The overall goal of this proposal is to understand molecular mechanisms regulating insulin receptor signaling. Insulin receptor substrate 1 (IRS-1) is an important downstream effector of insulin receptor signaling that mediates cell metabolism and mitogenesis. Recently, the ubiquitin-proteasome degradation pathway has been shown to be involved in negatively regulating IRS-1- mediated signal transduction in cells following prolonged exposure to insulin. How the ubiquitin-proteasome degradation pathway is activated by insulin and causes IRS-1 turnover is not completely understood. Our experiments will focus on defining the molecular mechanism of the insulin- induced ubiquitination of IRS-1, a key step in the proteasomal degradation pathway. Based on our preliminary data that indicated a role of insulin-stimulated E3 ligase activity in the ubiquitination of IRS-1 in vivo and in vitro, the proposed experiments will identify the specific E3 ligase for IRS-1 and define key biochemical steps in the ubiquitination of IRS-1. The specific aims are to: (1) Purify the E3 ligase for IRS-1 by affinity chromatography or Fast Performance Liquid Chromatography. The specific E3 ligase will be cloned and its role in the insulin-induced ubiquitination of IRS-1 will further defined; (2) Define the structural elements in IRS-1 essential for its specific ubiquitination, including ubiquitination sites, serine phosphorylation sites and domain that required for IRS-1 degradation; (3) Ascertain the role of PI3-kinase in insulin-induced ubiquitination of IRS-1. We anticipate that our studies will provide novel insights essential to understanding the role of IRS-1 turnover in regulating insulin receptor signal transduction and suggest new therapeutic strategies for treatment of Type 2 diabetes mellitus. [unreadable] [unreadable] [unreadable]